The bacterial restriction modification system consists of restriction endonucleases (restriction enzymes) and DNA methyltransferases, and the former are able to specifically recognize and cleave DNA while the latter are able to add a methyl modification to a base of DNA to prevent the cleavage of DNA by restriction enzymes. The Restriction modification system is able to selectively degrade exogenous DNA invading into bacteria to enable self-protection of bacteria. Restriction modification systems are divided into four major types, according to their subunit constitutions, cleavage sites, sequence specificities, and co-factor characteristics. Subunits of restriction enzymes of the restriction modification systems type I, type II, and type III are able to recognize and cleave non-methylated DNA. However, if DNA is first recognized and modified by the subunit of the methyltransferase, cleavage cannot be achieved by the restriction enzyme. The restriction modification system type IV consists of only the restriction enzymes and does not contain the methyltransferases. It recognizes and cleaves DNA having an exogenous methylation pattern, and thus is a methylation-dependent restriction enzyme. Additionally, a recent study indicates that phosphorothioation-modification-enzymes for DNA backbones and corresponding restriction enzymes thereof are a new type of restriction modification systems (Nucleic Acids Research, 38, 7133-7141). Such a complex modification-cleavage mode considerably protects the safety of bacteria's own DNA and is used as a main means of bacteria for effectively preventing invasion of exogenous DNA released by bacteriophages and dead bacteria in the environment. Meanwhile, this also becomes the main barrier for introducing an exogenous DNA into bacteria and enabling genetic manipulation by using molecular biological methods. The genetic manipulation of bacteria having multiple restriction modification systems is especially difficult.
To date, investigators have invented two types of techniques for overcoming the restriction modification barriers. The first strategy is modifying exogenous DNAs, including in vitro modification and in vivo modification of E. coli. For example, in vitro modification of exogenous DNAs using crude protein extract (containing DNA methyltransferase) of the target bacterium enables transformation of Helicobacter pylori, Bacillus cereus, and Bacillus weihenstephanensis (Molecular Microbiology, 37, 1066-1074, Applied and Environmental Microbiology, 74, 7817-7820); or cloning and expression of DNA methyltransferase of the target bacterium in E. coli and in vivo modification of exogenous plasmid DNAs, for example, cloning and expression of two DNA methyltransferases of Bifidobacterium adolescentis in E. coli TOP10 and modification of shuttle plasmids, enables genetic transformation of Bifidobacterium adolescentis (Nucleic Acids Research, 37, e3). The second type, i.e., a method for inactivating restriction modification systems, includes inactivation by physical means and gene knockout. By transitorily inactivating restriction enzymes of the target bacterium using heating after transformation, the transformation efficiency of exogenous plasmids for Corynebacterium glutamicum is increased to 108 CFU/μg DNA (Microbial Biotechnology, 52, 541-545); after gene CAC1502 is knocked out, Clostridium acetobutylicum can be allowed to accept unmethylated plasmid DNA (PLoS ONE, 5, e9038); and however, there is also a report indicating that knockout of Saul restriction endonuclease is not sufficient to allow Staphylococcus aureus to accept exogenous DNAs (Applied and Environmental Microbiology, 75, 3034-3038).
Although the techniques described above may increase transformation efficiency of exogenous DNAs for target bacterium to some extent, there are still the following problems and deficiencies: Although some solutions are able to perform in vitro modification on exogenous DNA molecules using DNA methyltransferases of the target bacterium, in vitro modification efficiency using the crude protein extract is low, and a part of plasmids would be also degraded at the same time of modification; the solution of in vivo modification has not eliminated methylation of exogenous DNA molecules by E. coli's own DNA methyltransferases, and such DNAs having an E. coli methylation pattern are prone to activate the restriction system type III of the target bacterium. Restriction enzymes of a number of bacteria are not heat-sensitive and cannot be transitorily inactivated by heating; if a bacterium contains multiple restriction modification systems, a lot of time and effort are needed to knockout restriction enzyme genes one by one, and meanwhile, knockout of restriction enzymes also causes the target bacterium to be infected by bacteriophages, which is extremely adverse to the construction of strains for industrial microbial fermentation. The techniques have poor generality and may be only applicable to one or a few types of bacteria.